Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/20011—Rhabdoviridae
  • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
  • C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to analogues of anti- thrombin polypeptides and to a process for their preparation.
• the analogues have been obtained by C-terminal modification of anti-thrombin polypeptides from the leech Hirudinaria manillensis and have an improved biological activity in comparison to the polypeptides from which they are derived.
• Hirudin originally isolated from the medicinal leech, Hirudo medicinalis. is a well known and well characterized polypeptidic inhibitor of thrombin 1 - 2 . More particularly, it binds thrombin by ionic interactions thus preventing the cleavage of fibrinogen to fibrin and the subsequent fibrin- clot formation.
• hirudin has demonstrated efficacy in preventing venous thrombosis, vascular shunt occlusion and thro bin-induced disseminated intravascular coagulation. In addition, hirudin exhibits low toxicity, little or no antigenicity and a very short clearance time from circulation. 3
• Polypeptides with anticoagulant properties have been isolated from a different leech species, Hirudinaria manillensis (EP-A-0347376 and WO 90/05143) .
• This leech is evolutionarily more advanced than Hirudo medicinalis and could therefore synthesize anticoagulant peptides whose amino acid sequences may be different from those of hirudin and other known hirudin variants.
• P,. 63 -Xaa-Z (I) wherein P, . ⁇ is the amino acid sequence from position 1 to position 63 of the polypeptide PI (SEQ ID NO: 1) or P2 (SEQ ID NO: 2) , Xaa is any amino acid residue and Z is -OH, -NH 2 , -Gly-OH or a dipeptide consisting of: a glycine or a polar amino acid residue, followed by an amino acid selected from the group consisting of: alanine, glycine, valine, leucine, isoleucine, proline, cysteine, methionine, serine, threonine, lysine, arginine, histidine, asparagine, glutamine, aspartic acid and gluta ic acid, with the proviso that when Z is -OH, -NH 2 or -Gly-OH, Xaa can not be asparagine and a pharmaceutically acceptable salt thereof.
• the pharmaceutically acceptable salts of the polypeptides may be acid addition salts. They may be salts with an inorganic acid such as a hydrohalic acid, such as hydrochloric acid; sulphuric acid; phosphoric acid; or pyrophosphoric acid.
• the salts may be salts with an organic acid such as benzenesulphonic, p-toluenesulphonic, methanesulphonic, acetic, lactic, palmitic, stearic, malic, tartaric, ascorbic or citric acid.
• the analogues also contain free carboxyl groups and may therefore be present as sodium, calcium, potassium, magnesium or ammonium salts or salts with a physiologically tolerable organic nitrogen-containing base.
• the analogues can also be in the form of inner salts.
• the polypeptides of the invention are able to inhibit the proteolytic effects of thrombin, for example fibrin formation from fibrinogen and the activation of factor V and factor VIII.
• the polypeptides are therefore useful, for example, as anticoagulant and antithrombotic drugs, for the treatment of thromboembolic pathologies such as disseminate intravascular coagulation, cerebral embolisms, deep venous thrombosis and arterial thrombosis. They are also useful in prophylactic treatment of coagulation disorders, for example for the therapy of acute myocardial infarction.
• the polypeptides of the invention consist essentially of the sequence P, .M -Xaa-Z. However, the polypeptides may be preceded by all or part, typically a C-terminal part, of a leader sequence.
• the leader sequence may be a native leader sequence of a natural polypeptide from which the polypeptides of the invention are derived, or may be a foreign leader sequence.
• the leader sequence may be capable of directing secretion of the polypeptides of the invention. Two of the natural anti-thrombin polypeptides are expressed with a leader sequence which is cleaved subsequently. All or part of this leader sequence may therefore . be present in the polypeptide of the invention, the sequence being: Met Phe Ser Leu Lys Leu Phe Val Val Phe Leu Ala Val Cys lie Cys Val Ser Gin Ala (SEQ ID NO: 32) .
• a polypeptide according to the invention, or a salt thereof, may be prepared by: 1) isolating an anti-thrombin polypeptide from the tissue or secretions of a leech of the species Hirudinaria manillensis and 2) subsequently modifying the C-terminal end of such a polypeptide. More specifically, the polypeptide can be obtained by obtaining a preparation of leech extract, subjecting the preparation to high pressure liquid chromatography, and modifying the C-terminal end of the polypeptide.
• a polypeptide according to the invention or a salt thereof can then be prepared by:
• a host in which a polypeptide according to the invention is able to be expressed is prepared by transforming a host with a compatible expression vector of the invention.
• the expression vector can be prepared by:
• an expression vector can be prepared by: (a) producing and isolating a cDNA encoding the polypeptide PI or P2 from mRNA of a leech of the species Hirudinaria manillensis;
• a polypeptide according to the invention is consequently prepared by providing a transformed host under such conditions that the polypeptide is expressed.
• the polypeptide can be obtained glycosylated.
• the polypeptide can be isolated as such or in the form of a pharmaceutically acceptable salt. In this way, a polypeptide or salt according to the invention may be obtained in essentially pure form.
• polypeptides of the invention may be modified by way of amino acid extension at either or each end.
• a polypeptide composed of such an extended sequence must of course still exhibit anti-thrombin activity.
• a short sequence of up to 30 amino acid residues may be provided at either or each terminus.
• polypeptides of the invention may be subjected to one or more post-translational modification such as sulphation, COOH- amidation, acylation or chemical alteration of the polypeptide chain.
• a polypeptide having a glycine residue at its carboxy terminus may be subjected to enzymatic amidation with peptidyl-glycine ⁇ -amidating monooxygenase (PAM enzyme) .
• PAM enzyme catalyzes the C- terminal oxydative cleavage of glycine-extended peptides to give the corresponding amidated product and glyoxylate 4 .
• the polypeptide P, .63 -Val-NH 2 constitutes a particular embodiment of the present invention.
• P[ ⁇ 3 -Val-NH 2 is the polypeptide of formula (I) wherein Xaa is Val and Z is -NH 2 .
• the polypeptide may be obtained by amidating the polypeptide of formula P ⁇ -Val-Gly-OH.
• the C-terminal -amide structure contributes to the biological stability of the amidated polypeptide conferring protection from the carboxypeptidase-mediated degradation, increases its in vivo half-life and confers a more selective receptor binding 5 .
• ⁇ -amidation represents an important and characteristic post-translational processing of many secreted peptides in eukaryotic organisms.
• the amidating activity of PAM enzyme has been shown to be dependent upon the presence of molecular oxygen and copper ions and to be stimulated by cofactors such as ascorbate, catalase or potassium iodide 6 .
• carboxypeptidase Y and, more generally the class of carboxypeptidases has been proved to be an efficient amidating enzyme 7 .
• carboxypeptidases are able to amidate polypeptides having any amino acid residue at their C-terminal end.
• the invention includes DNA sequences consisting essentially of the following sequences:
• the DNA coding sequence typically does not include introns.
• the DNA sequence is isolated and purified.
• the gene is inserted in an expression vector able to drive production of the recombinant product.
• the DNA sequence may be a mutagenized cDNA sequence.
• the DNA sequence may be a synthetic DNA sequence.
• the synthetic gene is typically prepared by chemically synthesising oligonucleotides which, in total, correspond to the desired gene. The oligonucleotides are then assembled to obtain the gene.
• a gene may therefore be constructed from six chemically synthesised oligonucleotides, each oligonucleotide representing about one third of one strand of a double- stranded DNA gene. The oligonucleotides are ligated and annealed to obtain the desired gene.
• a gene is constructed with restriction sites at each end to facilitate its subsequent manipulation.
• a DNA sequence may be provided which further encodes a leader peptide as mentioned above.
• the leader peptide is capable of directing secretion of the polypeptide from cells in which the polypeptide is to be expressed.
• the sequence encoding the leader peptide is typically fused to the 5'-end of the DNA sequence encoding the polypeptide.
• the leader peptide may be the OmpA leader peptide when expression in a bacterial host, such as E. coli is required.
• the leader peptide may be the leader peptide of vesicular stomatitis virus G protein (VSV G protein) when expression is to be in insect cells.
• VSV G protein vesicular stomatitis virus G protein
• Appropriate DNA sequences encoding the OmpA and VSV G protein leader sequences "are: OmpA leader: ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT 42 TTC GCT ACC GTA GCG CAG GCC (SEQ ID NO: 4) 63
• VSV G protein leader
• a DNA sequence may be provided which encodes a fusion protein which is cleavable to release a polypeptide of the invention.
• a DNA sequence may be used which encodes a carrier polypeptide sequence fused via a cleavable linkage to the N-terminus of a polypeptide of the invention.
• the cleavable linkage may be one cleavable by cyanogen bromide.
• an expression vector is constructed which comprises a DNA sequence encoding the polypeptide and which is capable of expressing the polypeptide when provided in a suitable host.
• Appropriate transcriptional and translational control elements are provided, including a promoter for the DNA sequence, a transcriptional termination site, and translational start and stop codons.
• the DNA sequence is provided in the correct frame such as to enable expression of the polypeptides to occur in a host compatible with the vector.
• the expression vector typically comprises an origin of replication and, if desired, a selectable marker gene such as an antibiotic resistance gene.
• a promoter is operably linked to the DNA sequence encoding the polypeptide.
• the expression vector may be a plasmid. In that case, preferably a promoter selected from the P l ⁇ , and P
• the expression vector may be a virus.
• the virus may be a recombinant baculovirus in which the polyhedrin promoter is operably linked to the DNA sequence encoding the polypeptide.
• An expression vector capable of expressing the polypeptide may be prepared in any convenient fashion.
• a DNA fragment encoding the polypeptide may be inserted into an appropriate restriction site of an expression vector, for example a plasmid vector.
• a recombinant baculovirus may be prepared by:
• step (i) cloning a gene encoding the polypeptide into a baculovirus transfer vector at a restriction site downstream of the polyhedrin promoter; and ( ⁇ ) co-transfecting insect cells susceptible to baculovirus infection with the recombinant transfer vector from step (i) and intact wild-type baculovirus DNA.
• the baculovirus transfer vector may be one having a unique cloning site downstream of the polyhedrin ATG start codon.
• the product that is then expressed by the resulting recombinant baculovirus will be a fusion protein in which a N-terminal portion of the polyhedrin protein is fused to the N-terminus of the polypeptide of the invention.
• a cleavable linkage may be provided at the fusion junction.
• the insect cells employed in step (ii) are typically Spodoptera fru iperda cells.
• the wild-type baculovirus is typically Autographa californica nuclearpolyhedrosis virus (AcNPV) .
• An expression vector encoding the polypeptide is provided in an appropriate host.
• Cells are transformed with the gene of the polypeptide.
• a transformed host is provided under such conditions that the polypeptide is expressed.
• Transformed cells for example, are cultivated so as to enable expression to occur. Any compatible host-vector system may be employed.
• the transformed host may be a prokaryotic or eukaryotic host.
• a bacterial or yeast host may be employed, for example E. coli or S. cerevisiae. Gram positive bacteria may be employed.
• a preferred bacterial host is a strain of E. coli type B.
• Insect cells can alternatively be used, in which case a baculovirus expression system is appropriate.
• the insect cells are typically Spodoptera fruqiperda cells.
• cells of a mammalian cell line may be transformed.
• a transgenic animal for example a non-human mammal, may be provided in which the polypeptide is produced.
• polypeptide that is expressed may be isolated and purified.
• a polypeptide having the aminoarid sequence depicted in formula (I) above preceded by a Met residue attributable to a translation start codon can be obtained.
• a fusion protein may be obtained comprising the amino acid sequence of formula (I) fused to a carrier sequence.
• the carrier sequence is tipically fused to the N-terminus of the polypeptide of formula (I) .
• a polypeptide having an amino acid sequence according to formula (I) can be released by cleavage with a suitable agent.
• a polypeptide of the invention or a pharmaceutically acceptable salt thereof can also be prepared by: (a) chemically synthesising the said polypeptide;
• polypeptides can therefore be built up by chemical synthesis from single amino acids and/or preformed peptides of two or more amino acids in the order of the sequence of the desired polypeptide. Solid-phase or solution methods may be employed. The resultant polypeptide may be converted into a pharmaceutically acceptable salt if desired.
• the amino acid sequence of the desired polypeptide is built up sequentially from the C- terminal amino acid which is bound to an insoluble resin.
• the desired polypeptide When the desired polypeptide has been produced, it is cleaved from the resin. When solution-phase synthesis is employed, the desired polypeptide may again be built up from the C- terminal amino acid. The carboxy group of this acid remains blocked throughout by a suitable protecting group, which is removed at the end of the synthesis.
• each amino acid added to the reaction system typically has a protected amino group and an activated carboxy group.
• Functional side-chain groups are protected too. After each step in the synthesis, the amino-protecting group is removed. Side-chain functional groups are generally removed at the end of the synthesis.
• a polypeptide may be converted into a pharmaceutically acceptable salt. It may be converted into an acid addition salt with an organic or inorganic acid.
• Suitable acids include acetic, succinic and hydrochloric acid.
• the polypeptide may be converted into a carboxylic acid salt such as the ammonium salt or an alkali metal salt such as the sodium or potassium salt.
• a polypeptide or pharmaceutically acceptable salt thereof may be used in a pharmaceutical composition, together with a pharmaceutically acceptable carrier or excipient therefor.
• Such a formulation is typically for intravenous administration (in which case the carrier is generally sterile saline or water of acceptable purity) .
• a polypeptide according to the invention is an anti-thrombin polypeptide and is suitable for treatment of thromboembolic events, such as the coagulation of blood, typically in a human patient.
• a polypeptide according to the present invention displays improved biological characteristics when compared to the corresponding native polypeptide from which it is derived.
• a polypeptide can therefore be used for the therapy and prophylaxis of thromboses and thromboembolisms, including the prophylaxis of post-operative thromboses, for acute shock therapy (for example for septic or polytraumatic shock) , for the therapy of consumption coagulopathies, in haemodialyses, haemoseparations and in extracorporeal blood circulation.
• the polypeptide or salt thereof can be coadministered with a plasminogen activator, such as tissue plasminogen activator.
• the dosage depends especially on the specific form of administration and on the purpose of the therapy or prophylaxis.
• the size of the individual doses nd the administration regime can best be determined by way of an individual judgement of the particular case of illness; the methods of determining relevant blood factors required for this purpose are familiar to the person skilled in the art.
• the therapeutically effective amount of the compounds according to the invention is in a dosage range of from approximately 0.005 to approximately 0.1 mg/kg body weight. A range of from approximately 0.01 to approximately 0.05 mg/kg body weight is preferred.
• the administration is effected by intravenous, intramuscular or subcutaneous injection.
• compositions for parenteral administration in single dose form contain per dose, depending on the mode of administration, from approximately 0.4 to approximately 7.5 mg of the compound according to the invention.
• these pharmaceutical compositions usually also contain a buffer, for example a phosphate buffer, which is intended to keep the pH value between approximately 3.5 and 7, and also sodium chloride, mannitol or sorbitol for adjusting the isotonicity.
• a buffer for example a phosphate buffer, which is intended to keep the pH value between approximately 3.5 and 7, and also sodium chloride, mannitol or sorbitol for adjusting the isotonicity.
• They may be in freeze-dried or dissolved form, it being possible for solutions advantageously to contain an antibacterially active preservative, for example from 0.2 to 0.3% 4-hydroxybenzoic acid methyl ester or ethyl ester.
• a composition for topical application can be in the form of an aqueous solution, lotion or gel, an oily solution or suspension or a fat-containing or, especially, emulsified ointment.
• a composition in the form of an aqueous solution is obtained, for example, by dissolving the active ingredients according to the invention, or a therapeutically acceptable salt thereof, in an aqueous buffer solution of from e.g., pH 4 to pH 6.5 and, if desired, adding a further active ingredient, for example an anti-inflammatory agent,and/or a polymeric binder, for example polyvinylpyrrolidone, and/or a preservative.
• the concentration of active ingredient is from approximately 0.1 to approximately 1.5mg, preferably from 0.25 to 1.0 mg, in 10 ml of a solution or 10 g of a gel.
• An oily form of administration for topical application is obtained, for example, by suspending the active ingredient according to the invention, or a therapeutically acceptable salt thereof, in an oil, optionally with the addition of swelling agents, such as aluminium stearate, and/or surfactants (tensides) having an HLB value ("hydrophilic- lipophilic balance") of below 10, such as fatty acid monoesters of polyhydric alcohols, for example glycerin monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate.
• a fat-containing ointment is obtained, for example, by suspending the active ingredient according to the invention, or a salt thereof, in a spreadable fatty base, optionally with the addition of a tenside having an HLB value of below 10.
• An emulsified ointment is obtained by triturating an aqueous solution of the active ingredient according to the invention, or a salt thereof, in a soft, spreadable fatty base with the addition of a tenside having an HLB value of below 10. All these forms for topical application can also contain preservatives.
• the concentration of active ingredient is from approximately 0.1 to approximately 1.5mg, preferably from 0.25 to 1.0 mg, in approximately 10 g of base.
• compositions described above and pharmaceutical compositions analogous thereto that are intended for direct medicinal use in the body of a human or a mammal relate also to pharmaceutical compositions and preparations for medicinal use outside the living body of humans or mammals.
• Such compositions and preparations are used especially as anticoagulant additives to blood that is being subjected to circulation or treatment outside the body (for example extra corporeal circulation or dialysis in artificial kidneys) , preservation or modification (for example haemoseparation) .
• preparations such as stock solutions or alternatively preparations in single dose form, are similar in composition to the injection preparations described above; however, the amount or concentration of active ingredient is advantageously based on the volume of blood to be treated or, more precisely, on its thrombin content.
• PI to P3 denote the three different peaks obtained from the preparation according to Example 1, FT is for flow through and 4-AB is for 4- aminobenzamidine.
• Figure 2 shows the elution profiles obtained in Example 2(b) for trypsin-digested PE-P1 (A) and PE-P2 (B) .
• Figure 3 shows the nucleotide sequence of the six oligos coding for the analogue P2,. 63 -Val-Gly-OH linked to the OmpA leader peptide.
• P2,. 63 -Val-Gly-OH is an analogue of Formula (I) wherein P is the anti-thrombin polypeptide P2, Xaa is Val and Z is Gly-OH.
• the sequence shown in bold face indicates the OmpA leader peptide; the Hind III, Bal I and BamHI restriction sites are underlined and the codons for Val ⁇ and Gly 6S are in italics.
• Figure 4 shows the scheme of the construction of the plasmid, named 0MP-P2VG, which is the source of a Bal I-BamHI DNA fragment for further genetic constructions.
• Figure 5 shows schematically the construction of pFC- P2VG which is the plasmid used for the production of P2,. 63 - Val-Gly-OH protein in E. coli.
• Figure 6 shows the general structure of the plasmid pOMPA-P2VG used for the production of P2,. 63 -Val-Gly-OH in E. coli.
• Figure 7 shows the nucleotide sequence and assembling of the synthetic oligos used for the secretion of P2,. 63 -Val-Gly- OH from insect cells. The sequence shown in bold face indicates the VSV G protein leader peptide.
• FIG 8 is a schematic representation of the construction of a new recombinant M13, named VSV-P2VG, where the complete P2,_ 63 -Val-Gly-OH gene is linked to the VSV G protein leader peptide.
• Figure 9 shows schematically the construction of pAc- P2VG which has been used as transfer vector to the baculovirus genome.
• pAcYMl is the starting plasmid widely used as acceptor of heterologous sequences to be transferred to the virus.
• Figure 10 shows the nucleotide sequence and assembling of the synthetic oligos coding for the beginning of the P2,. 63 Val-Gly-OH chain. The ATG codon coding for the additional methionine residue is shown in bold face.
• Figure 11 shows schematically the construction of pAcFTl, which has been used for intracellular expression.
• Figure 12 is a schematic representation of a new transfer plasmid, named pAcFTl-P2VG, which carries the complete P2,. 63 - Val-Gly-OH sequence linked to the first 18 amino acids of polyhedrin. This plasmid has been used to transfer the heterologous sequence to the baculovirus genome.
• Figure 13 shows the nucleotide sequence of the six oligos coding for the analogue Pl ⁇ -Val-Gly-OH linked to the OmpA leader peptide.
• the sequence shown in bold face indicates the OmpA leader peptide; the Hind III, Bal I and BamHI restriction sites are underlined and the codons for Val M and Gly 6S are in italics.
• Example 1 Example 1
• An antithrombin preparation was prepared from Hirudinaria manillensis leeches according to the procedure illustrated under a) to d) below: a) Acetone extraction Ethanol dried leech heads (2920 g) were finely chopped into small pieces and treated with a mixture 40:60 acetone/water (7.5 1) . After homogenisation with stirring at room temperature, the mixture was spun for 15 min at 2,700 rpm and the supernatant was decanted; the pellet was again resuspended in 40:60 acetone:water mixture, stirred for 30 min and the mixture centrifuged for 15 min at 2,700rpm. The supernatant was pooled with the initial one and acidified to pH 4.5 with glacial acetic acid (vol. 8.5 1) .
• the mixture was spun at 2,700 rpm for 15 minutes, then the supernatant was decanted and the pH of the solution adjusted to pH 6.0 by adding 30% ammonia. Following rotary evaporation at 35°C, the pH of the concentrated solution was lowered to 1.8; precipitated contaminants were removed by centrifuging and the raw anti- thrombin material was precipitated from the mixture using a 9-fold acetone excess. The mixture was then spun down, the pellet resuspended in acetone and again centrifuged. The precipitated material was then lyophilized. b) Ionic exchange purification The raw anti-thrombin material was reconstituted in water, dialyzed against 10 mM ammonium acetate buffer at pH
• CMSepharose Pharmacia, 2.6 x 30 cm
• anti-thrombin active fractions were eluted with 20mM ammonium acetate pH 4.5, collected and pooled (1.3 1) .
• pooled fractions were concentrated to 0.5 1 in a Minitan apparatus (Millipore) ; the concentrated solution was neutralised with NaOH and then applied on to a Q Sepharose column equilibrated in 20 mM Tris-HCl pH 7.0. The bound material was eluted with a linear gradient of 0 - 1 M NaCl in the starting buffer.
• the fractions containing with anti-thrombin activity were pooled, concentrated and desalted on a Superdex S-200 column eluted with 20 mM Tris-HCl pH 7.5 at a flow rate of 4ml/min. Active pool from gel filtration was concentrated by Minitan and further purified by weak anion exchange chromatography (DEAE FPLC) . The active material was loaded onto a Protein Pak DEAE-5PW column (Waters) and eluted with a gradient of 0 - 1 M NaCl in 20 mM Tris-HCl pH 6.5, at a flow rate of 1.0 ml/min. Active fractions were pooled, characterized for protein content and activity (specific activity: 800 ATU/mg), and freeze-dried in a Speed Vacconcentrator (Savant) .
• Thrombin-Sepharose Commercial bovine thrombin (Sigma) was further purified according to the procedure described by Lundblad 9 and then was attached to activated Sepharose CL 6B (Pharmacia) following manufacturer's instructions.
• the column (1.7 ml) was equilibrated with 50 mM Tris-HCl pH 8.3 and the freeze- dried material from DEAE-FPLC (reconstituted in buffer) was loaded.
• the column was subjected to three washings, in starting buffer, then in the same buffer containing 3.0 M NaCl and again with starting buffer (each washing was three times the column volume). Flow rate was 0.3 ml/min.
• the bound material was eluted with 10 ml of 0.1M 4- aminobenzamidine in 25 mM HC1.
• the active fractions were pooled and buffer exchanged in 50 mM Tris-HCl pH 8.3 onto a PD-10 column (Pharmacia) .
• Peaks of protein (detected at 220 nm) were manually collected, concentrated under vacuum and re-chromatographed under the same conditions.
• PE-P1 and PE-P2 Trypsin digestion and peptide mapping of PE-P1 and PE-P2 - Purified PE-P1 and PE-P2 (respectively 10 and 20 ⁇ g) were digested with TPCK-treated trypsin (Sigma) in 200 ⁇ l of 1% ammonium bicarbonate pH 8.0 in the presence of
• Trypsin was added at an enzyme-to- substrate ratio of 1:20 (w/w) and incubation was carried out for 4 hours at 37°C.°C. Digestion was stopped by freeze- drying in Savant.
• the nucleotide sequence coding for the analogue P2,. 63 - Val-Gly-OH was designed on the basis of the Escherichia coli preferred codons". Moreover, a Ball restriction site was engineered very close to the 5' end of the synthetic P2, ⁇ 3 - Val-Gly-OH coding sequence to allow insertion of such sequence in different expression vectors. Indeed, the same synthetic gene was used for expression of recombinant P2,. 63 - Val-Gly-OH protein in bacterial and insect cells. All plasmid DNA manipulations were carried out as described by Maniatis et al 12 . In the case of insect cells methods were developed which yielded protein P2,.
• 63 -Val-Gly-OH as a secreted or cytoplasmic product.
• methods were developed to obtain secretion to the periplasm of the recombinant product.
• it is necessary to synthesize the P2,.
• VSV Vescicular Stomatitis Virus
• Gly-OH gene for the expression vectors used in the Examples six synthetic complementary oligonucleotides were synthetized using an automated DNA synthetizer (Applied Biosystems) and their sequence is shown in Figure 3.
• oligos 1 and 2 Their sequence, shown in Fig.. 3 as oligos 1 and 2, includes also the beginning of the P2,. 63 -Val-Gly-OH gene coding for the first 10 amino acids. Following enzymatic phosphorylation the six oligos were assembled using DNA ligase and the resulting double-strand sequence was inserted in the M13 phage vector mpl8, (Yanisch-Perron et al.. Gene
• the P2, .63 -Val-Gly-OH gene can be excised as a Hindlll-BamHI fragment which codes for the OmpA Shine- Dalgarno and leader peptide followed by the P2,. 63 -Val-Gly-OH coding sequence.
• This restriction fragment is now ready to be inserted in an appropriate expression vector.
• Several expression systems could, theoretically, be err.ployed to obtain high level production of heterologous proteins in bacteria. The system based on the promoter P, has been used with success in our laboratory in the past 19 . Again, even in the case of the selected promoter, the levels of expression of a given polypeptide cannot be predicted.
• Plasmid pFC33 shown in Figure 5, has already been described in the literature 19 . It carries the resistance to the antibiotic ampicillin and the bacterial promoter P ⁇ which drives expression of proapolipoprotein Al. Following digestion of pFC33 with Hindlll and BamHI, the large Hindlll- BamHI fragment, carrying the antibiotic resistance gene and the promoter, was isolated and joined to the Hindlll-BamHI fragment from OMP-P2VG coding for the P2,. 63 -Val-Gly-OH gene. The details of this construction are shown in Figure 5. We isolated a new plasmid, named pFC-P2VG, which is the final plasmid for the production of P2,. 61 -Val-Gly-OH in E. coli.
• An object of the present invention is the use of Ji. coli strains of the type B for the expression and secretion to the periplasm of P2,. 63 -Val-Gly-OH and the other anti-thrombin analogues of the invention. Indeed, we have found that insertion of plasmid pFC-P2VG in type B strains of the bacterium E. coli brings high level production of P2 l . 63 -Val- Gly-OH. Interestingly, different strain types of E. coli do not work as efficiently and it seems, therefore, that the host strain type is crucial for the successful production of P2,. 63 -Val-Gly-OH.
• strains of E. coli are available and can be used for the production of P2,. 63 -Val-Gly-OH.
• Preferred strains are ATCC 12407, ATCC 11303, NCTC 10537.
• Competent cells of strain NCTC 10537 were prepared using the calcium chloride procedure of Mandel and Higa 21 . Approximately 200 ⁇ l of a preparation of these cells at 1 x 10 9 cells per milliliter were transformed with 2 ⁇ l of plasmid DNA (approximate concentration 5 ⁇ g/ml) . Transformants were selected on plates of L-agar containing lOO ⁇ g/ml ampicillin.
• Example 5 Expression and secretion of protein P2 ] ⁇ -Val-Gly- OH from insect cells
• the synthetic Hindlll-Ball fragment was joined to apurified Ball-BamHI fragment from OMP-P2VG carrying the
• VSV-P2VG P2, ⁇ 3 -Val-Gly-OH gene and inserted in M13mpl8 previously cut with Hindlll and BamHI.
• This construction which yielded a new plasmid named VSV-P2VG is schematically shown in Figure 8. From VSV-P2VG we have excised a BamHI-BamHI DNA fragment carrying the P2,. 63 -Val-Gly-OH gene fused to tne VSV leader peptide which was then inserted into the vector pAcYMl 25 , as shown in Figure 9. The resulting plasmid was named pAc-P2VG.
• the VSV-P2VG coding sequence must be transferred to the baculovirus genome under the transcriptional control of the polyhedrin promoter.
• insect cells As insect cells, Spodoptera frugiperda cells were chosen as host cells. Experimental details are as follows: S. frugiperda cells were transfected with a mixture of infectious AcNPV DNA and plasmid DNA representing the individual recombinant transfer vectors by a modification of the procedure described by Summers et al 26 .
• One microgram of viral DNA was mixed with 25-100 ⁇ g of plasmid DNA and precipitated with (final concentrations) 0.125 M calcium chloride in the presence of 20 mM HEPES buffer, pH 7.5, 1 mM disodium hydrogen orthophosphate, 5mM potassium chloride, 140 mM sodium chloride and 10 mM glucose (total volume 1ml) .
• the DNA suspension was inoculated onto a monolayer of 10 6 S. frugiperda cells in a 35-mm tissue culture dish, allowed to adsorb to the cells for 1 h at room temperature, then replaced with 1 ml of medium. After incubation at 28°C for 3 days the supernatant fluids were harvested and used to produce plaques in S. frugiperda cell monolayers. Plaques containing recombinant virus were identified by their lack of polyhedra when examined by light microscopy. Virus from such plaques was recovered and after further plaque purification was used to produce polyhedrin-negative virus stocks.
• Example 6 Expression of protein P2, 63 -Val-Glv-OH in the cytoplasm of insect cells
• Protein P2, .63 -Val-Gly-OH could also be produced and accumulated in the cytoplasm of S. frugiperda cells. This approach generally gives a better yield of heterologous proteins since it utilizes the expression signals of polyhedrin which is a non-secreted viral protein.
• the recombinant baculovirus was obtained as described in Example 5. Infection of S. frugiperda cells was carried out according to standard procedures 26 . Cultivation of infected insect cells leads to the cytoplas ic accumulation of the fusion protein. This hybrid protein was the source of recombinant protein P2 ⁇ -Val-Gly-OH.
• Several methods are available from the literature which can be used to cleave the hybrid with CNBr 28"29 .
• the application of the method of Olson et a_l has allowed us to obtain the correct polypeptidic sequence of P2 ! . 63 -Val-Gly-OH. This molecule displayed anti- thrombin activity.
• the analogue P, .63 -Val-Gly-OH is converted to the corresponding amidated product P,. 63 -Val-NH 2 by incubation with a partially purified protease-free PAM enzyme preparation, as, for example, obtained from rat thyroid medullary carcinoma 30 or from the conditioned medium of cells derived from the same tumour 31 .
• recombinant DNA techniques can also be applied to prepare an amidating enzyme.
• the enzymatic amidating reaction has been carried out in an aqueous buffer, for example TSE buffer, supplemented with copper ions, ascorbate, catalase, potassium iodide, SDS and
• the progress of the amidation reaction can be monitored by the method of Corbett and Corbett 32 which detects the formation of glyoxylic acid after derivatization with nitrosobenzene.
• the amidation product P,. 63 -Val-NH 2 can be purified according to standard techniques well known to the skilled in the art.
• Example 9 Biological activity of P2, 63 -Val-Gly-OH Antithrombin activity
• the antithrombin activity of the analogues P2 1 . 63 -Val-Gly-OH of the present invention was determined on the basis of the rapid and stoechiometric reaction of the analogue peptides with thrombin.
• Thrombin activity was measured quantitatively by means of titration of a standard solution of thrombin 33 .
• Thrombin activity was calibrated with the International Standard Preparation of Thrombin (c 70/157) obtained from the National Institute for Biological Standards and Control (London, U.K.) and expressed in National Institute of Health Units (N.I.H. units) .
• the thrombin neutralizing activity of the samples was expressed as antithrombin units (ATU) ; one ATU is the amount of test compound which neutralizes an NIH unit of thrombin.
• Recombinant hirudin (HVl variant; rHVl) and recombinant P2 protein (rP2) were considered as reference compounds.
• the test was performed as follows: 0.2 ml of a standard solution of 0.05% human fibrinogen (Kabi Vitrum, Sweden) in Tris HCl buffer pH 7.4 were incubated at 37°C in the presence of 0.01 to 0.1 ml of the solutions of P2 ⁇ -Val-Gly-OH.
• the end point of the titration was considered to be reached when a fibrin clot was formed within one minute. Indeed, clot formation only occurred when a sufficient amount of thrombin was added to the reaction mixture, the added thrombin being able to neutralize the total amount of the analogue present in the mixture.
• the antithrombin activity of the analogue P2,. 63 -Val-Gly-OH has been expressed in ATU/mg protein (see table 1) .
• Protein content was determined by aminoacid analysis. Chromogenic substrate assay
• the principle of the method is based on the inhibitory activity of P2, .63 -Val-Gly-OH on the reaction between thrombin and its specific chromogenic substrate S-2238 34 .
• the inhibition of the reaction between thrombin and the chromogenic substrate S-22? ⁇ was calculated as the ratio (expressed as percentage) between the mean ⁇ Abs/min. before and after the addition of the analogue. A standard curve has been established on the basis of the percent inhibition versus the concentration of the sample.
• the antithrombin activity of the analogue P2,. 63 -Val-Gly-OH has been determined by measuring the thrombin time in human plasma.
• Val-Gly-OH resulted considerably superior to that of the reference compounds.
• Table 1 reports the data obtained with the analogue P2,. 63 -Val-
• ORGANISM vesicular stomatitis virus
• ix FEATURE:
• TGGTTCTAAC GTTTGCGGTG AAGGTAAAAA CTGCCAGCTG TCTTCTTCTG 100 GTAACCAGTG CGTTCACGGT GAAGGTACCC CGAAACCGAA ATCTCAGACT 150
• ORGANISM vesicular stomatitis virus
• ORGANISM vesicular stomatitis virus
• ORGANISM baculovirus
• ORGANISM baculovirus

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Genetics & Genomics (AREA)
• General Chemical & Material Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Animal Behavior & Ethology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Public Health (AREA)
• Engineering & Computer Science (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Gastroenterology & Hepatology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Molecular Biology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Virology (AREA)
• Diabetes (AREA)
• Hematology (AREA)
• Peptides Or Proteins (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Laminated Bodies (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Saccharide Compounds (AREA)

Applications Claiming Priority (3)

Publications (2)

Family

ID=11366661

Family Applications (1)

Country Status (5)

Families Citing this family (3)

Family Cites Families (3)

• 1993
  • 1993-07-22 IT IT93MI001627A patent/IT1266561B1/it active IP Right Grant
• 1994
  • 1994-06-17 WO PCT/EP1994/001979 patent/WO1995003409A1/en not_active Ceased
  • 1994-06-17 JP JP7504879A patent/JPH09503646A/ja not_active Ceased
  • 1994-06-17 EP EP94920953A patent/EP0710285B1/de not_active Expired - Lifetime
  • 1994-06-17 DE DE69427084T patent/DE69427084T2/de not_active Expired - Fee Related

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events